Loss of ERBB2 and ERBB3 Receptors Impacts Epidermal Differentiation in Mice

Antibody-based cancer therapy has achieved significant success, and mAbs targeting ERBB2 (trastuzumab, pertuzumab) and ERBB3 (patritumab, seribantumab, lumretuzumab) are used to treat various types of cancer (Propper et al. , 2023Propper D. J. Gao F. Saunders M. P. Sarker D. Hartley J. A. Spanswick V. J. et al. Panther: AZD8931, inhibitor of EGFR, ERBB2 and ERBB3 signalling, combined with FOLFIRI: a Phase I/II study to determine the importance of schedule and activity in colorectal cancer. Br J Cancer. 2023; 128: 245-254Crossref PubMed Scopus (0) Google Scholar). The ERBB2/3 heterodimer, the most active signaling dimer in the ERBB family (Schneider and Yarden, 2016Schneider M. R. Yarden Y. The EGFR-HER2 module: a stem cell approach to understanding a prime target and driver of solid tumors. Oncogene. 2016; 35: 2949-2960Crossref PubMed Scopus (54) Google Scholar), is often hyperactive in tumors, making it a promising target in cancer therapy. Although antibodies targeting EGFR and their toxicity are largely recognized (Lichtenberger et al. , 2013Lichtenberger B. M. Gerber P. A. Holcmann M. Buhren B. A. Amberg N. Smolle V. et al. Epidermal EGFR controls cutaneous host defense and prevents inflammation. Sci Transl Med. 2013; 5199ra111Crossref PubMed Scopus (196) Google Scholar), the side effects of targeting the related Y kinase receptors ERBB2 and ERBB3 remain to be determined (Braden and Anadkat, 2016Braden R. L. Anadkat M. J. EGFR inhibitor-induced skin reactions: differentiating acneiform rash from superimposed bacterial infections. Support Care Cancer. 2016; 24: 3943-3950Crossref PubMed Scopus (31) Google Scholar; Hynes and Lane, 2005Hynes N. E. Lane H. A. ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer. 2005; 5: 341-354Crossref PubMed Scopus (2804) Google Scholar). The development of anticancer drugs depends on preclinical research based on reliable animal models able to predict the efficacy and toxicity of candidate drugs or drug combinations. Therefore, we generated a skin-specific double knockout for both receptors (Erbb2/3del) by crossing mice carrying conditional Erbb2 and Erbb3 alleles with transgenic mice expressing keratin (K) 5 promoter–driven cre recombinase. All animal experiments were approved by the governmental body of the state of Upper Bavaria, Germany and by the Austrian Federal Ministry of Education, Science, and Research. Previous studies with skin-specific knockouts of ERBB2 (Dahlhoff et al. , 2017bDahlhoff M. Muzumdar S. Schäfer M. Schneider M. R. ERBB2 is essential for the growth of chemically induced skin tumors in mice. J Invest Dermatol. 2017; 137: 921-930Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) or ERBB3 (Dahlhoff et al. , 2015Dahlhoff M. Schäfer M. Muzumdar S. Rose C. Schneider M. R. ERBB3 is required for tumor promotion in a mouse model of skin carcinogenesis. Mol Oncol. 2015; 9: 1825-1833Crossref PubMed Google Scholar) revealed that both proteins are dispensable for skin development and homeostasis but are required for the progression of skin tumors. Similarly, Erbb2/3del mice were born at the predicted ratios and showed no abnormalities in coat appearance or behavior but were reduced in body size and weight (Figure 1a). From the age of 3 months, Erbb2/3del mice showed skin changes and often developed a brittle, thickened epidermis on the tail and in the neck and abdomen area, where wounds with scabs occasionally appeared later (Figure 1b). These alterations were never seen in control littermates. Moreover, H CAR, acylcarnitine; CE, cholesterol ester; Cer, ceramide; DS, desmosterol sulfate; NAE, N-acylethanolamine; NDAE, N, N-diacylethanolamine; PCNA, proliferating cell nuclear antigen; TG, triglyceride. View Large Image Figure ViewerDownload Hi-res image Download (PPT) In light of the changes observed in sebaceous glands, we next extracted hair lipids (which are essentially of sebaceous origin) and assessed their composition by mass spectrometry. Lipidome investigations suggested a significant reduction of acylcarnitines, triglycerides, cholesterol esters, desmosterol sulfate, N-acylethanolamine, N, N-diacylethanolamine, and 2 subclasses of ceramides in the hair lipids of Erbb2/3del mice compared with those in the control littermates (Figure 2c). Thus, loss of ERBB2/3 increases sebaceous gland size but decreases the synthesis of specific lipids, which may influence the skin barrier. Because loss of ERBB2/3 alters epidermal differentiation, we set out to study the role of ERBB2 and ERBB3 receptors in the human HaCaT keratinocyte cell line. By employing CRISPR/Cas9 gene editing technology, we deleted ERBB2, ERBB3, and both receptors in HaCaT cells, thus expanding our previous studies (Dahlhoff et al. , 2017aDahlhoff M. Gaborit N. Bultmann S. Leonhardt H. Yarden Y. Schneider M. R. CRISPR-assisted receptor deletion reveals distinct roles for ERBB2 and ERBB3 in skin keratinocytes. FEBS J. 2017; 284: 3339-3349Crossref PubMed Scopus (0) Google Scholar) (Supplementary Figure S2a). All knockout clones were verified by western blot analysis (Supplementary Figure S2b). ERBB2/3del cells show that significantly increased levels of K5; K6; and K17 and the cornified layer proteins FLG, involucrin, and loricrin were increased but that cytoskeleton proteins ACTB and vinculin were significantly less expressed (Supplementary Figure S3). We in part confirmed these results in primary keratinocytes by small interfering RNA knockdown for ERBB2/3 for 4 (Supplementary Figure S4) and 6 (Supplementary Figure S5) days. In this study, as in HaCaT cells, EGFR phosphorylation is reduced, and loricrin expression is significantly increased. To evaluate the consequences of deleting ERBB2 and ERBB3 on gap closure, confluence formation, and velocity, all cell lines were analyzed with a lens-free live cell-imaging technology. A scratch assay showed significantly increased gap closure of ERBB2del cells and a significantly decreased gap closure behavior for ERBB3del and ERBB2/3del cells compared with those of control cells (Supplementary Figures S6a and S7). All clones showed a significantly decreased confluence formation compared with the control mock cell line (Supplementary Figure S6b). ERBB3del and ERBB2/3del cells were more affected than ERBB2del cells, suggesting that ERBB3 may play a greater role here than ERBB2. The velocity was significantly increased in all knockout cell lines compared with that in the controls (Supplementary Figure S6c). However, the data clearly show that the ERBB2del and the ERBB2/3del cells move much faster than the ERBB3del cells. Notably, only ERBB2/3del cells showed significant changes in all 3 assays, whereas the single knockouts showed no changes in at least 1 assay, indicating a cumulative or synergistic effect in the double knockout cells and resembling the results in triple knockouts for EGFR, ERBB2, and ERBB3 in canine kidney cells (Matsuda et al. , 2023Matsuda K. Hirayama D. Hino N. Kuno S. Sakaue-Sawano A. Miyawaki A. et al. Knockout of all ErbB-family genes delineates their roles in proliferation, survival and migration. J Cell Sci. 2023; 136jcs261199Crossref Scopus (0) Google Scholar). The interaction between the EGFR and adhesion proteins such as E-cadherin (CDH1) regulates contact inhibition of proliferation (Rübsam et al. , 2017Rübsam M. Mertz A. F. Kubo A. Marg S. Jüngst C. Goranci-Buzhala G. et al. E-cadherin integrates mechanotransduction and EGFR signaling to control junctional tissue polarization and tight junction positioning. Nat Commun. 2017; 8: 1250Crossref PubMed Scopus (138) Google Scholar). To investigate whether the loss of ERBB2 and ERBB3 changes contact inhibition, the phosphorylation of EGFR was analyzed in a confluence experiment. In mock cells as well as in the ERBB2del and ERBB3del cells, EGFR is significantly more phosphorylated in 100% confluent cells than in 50% confluent cells (Supplementary Figure S8a and b). However, in ERBB2/3del cells, EGFR is equally activated in 100% confluent cells and in 50% confluent cells (Supplementary Figure S8a and b). In mock, ERRB2del, and ERBB3del cells, CDH1 is significantly increased in 100% confluent cells compared with that in 50% confluent cells but not in double knockout cells, where the CDH1 signal is equally strong in 100 and 50% confluent cells (Supplementary Figure S8a and c). ERBB2 and ERBB3 appear to regulate the activation of EGFR in nonconfluent cells because the loss of ERBB2/3 leads to increased activation of EGFR, resulting in increased expression of CDH1, which is normally only seen in confluent cells. In summary, our data show that concomitant deletion of ERBB2 and ERBB3 in mice leads to impaired differentiation of the epidermis, inflammation, and altered sebaceous glands and eventually results in skin wound lesions. Although the single loss of ERBB2 or ERBB3 seems to be compensated by other ERBB components, this ability is lost upon a simultaneous deletion of ERBB2 and ERBB3. Inhibition of both ERBB2 and ERBB3 is thus an attractive approach for tumor therapies because both receptors are often active together in tumors, but their inhibition, similarly to the inhibition of EGFR, leads to cutaneous side effects. All animal experiments were approved by the governmental body of the state of Upper Bavaria, Germany (Regierung von Oberbayern, 55. 2-1-54-2532-206-2012) and by the Austrian Federal Ministry of Education, Science, and Research (BMBWF-68. 205/106-V/3b/2019) and were performed in strict compliance with the European Communities Council Directive (86/609/EEC) recommendations for the care and use of laboratory animals. All data generated or analyzed during this study are included in this published article and its supplementary information materials. Theresa Hommel: http: //orcid. org/0009-0009-0922-9948 Paula F. Meisel: http: //orcid. org/0009-0009-4657-2358 Emanuela Camera: http: //orcid. org/0000-0001-6633-0449 Grazia Bottillo: http: //orcid. org/0000-0002-6671-7384 Andrea R. Teufelberger: http: //orcid. org/0000-0002-1142-949X Theresa H. Benezeder: http: //orcid. org/0000-0001-6218-2792 Peter Wolf: http: //orcid. org/0000-0001-7777-9444 Lisa Kleissl: http: //orcid. org/0000-0001-8011-796X Georg Stary: http: //orcid. org/0000-0003-1746-4250 Christian Posch: http: //orcid. org/0000-0003-0296-3567 Marlon R. Schneider: http: //orcid. org/0000-0002-9570-3491 Maik Dahlhoff: http: //orcid. org/0000-0001-9189-7631 The authors state no conflict of interest. We thank Ingrid Renner-Müller and Petra Renner (Gene Center, Ludwig Maximilian University of Munich, Munich, Germany) for excellent animal care, Franziska Kress for assistance with western blot analysis, and Josef Millauer for mouse genotyping. We thank Angel Ramirez and Jose Jorcano (CIEMAT, Madrid, Spain) for providing K5-Cre mice and Carmen Birchmeier (Max-Delbrück-Center for Molecular Medicine, Berlin, Germany) for providing Erbb2-floxed mice. No funding was available for this study. Conceptualization: MD, MRS, TH; Data Curation: MD, TH, PFM, EC, THB, GB, ART, LK; Formal Analysis: MD, TH, PFM, EC, THB, GB, ART, CB, LK; Investigation: MD, TH, PFM, EC, GB, THB, CB, LK; Methodology: MD, TH, PW, EC, GS, CP; Project Administration: MD, MRS; Resources: MD, PW, GC; Visualization: MD, TH, MRS, EC, PW, GS; Writing – Original Draft Preparation: MD, TH, MRS, PW; Writing – Review and Editing: MD, TH, MRS, EC, GS, CP, PW Supplementary Table S1Antibodies Employed for Western Blot Analysis and Immunofluorescence StainingAntigenAntibodyHostDilutionEGFRCell Signaling Technology, number 2232Rabbit1: 1000ERBB2Cell Signaling Technology, number 4290Rabbit1: 1000ERBB2R IVL, involucrin; K, keratin; LOR, loricrin; p-AKT, phosphorylated protein kinase B; PCNA, proliferating cell nuclear antigen; p-ERBB, phosphorylated ERBB; p-EGFR, phosphorylated EGFR; p-P44/42 MAPK, phosphorylated P44/42 MAPK; VCL, vinculin. Open table in a new tab Supplementary Table S2List of Metabolites Determined by GC-MS in the Sebum Extract from Mouse HairSystematic NameSupplierSynonymsFormulaMWTMSQuantifier ionRT AvgTypeLabelDodecanoic acidLARODANC12: 0C12H24O2200. 21257. 216. 2EveneFATetradecanoic acidLARODANC14: 0C14H28O2228. 21285. 219. 7EveneFAHexadecanoic acidLARODANC16: 0C16H32O2256. 21313. 222. 9EveneFAOctadecanoic acidLARODANC18: 0C18H36O2284. 31341. 325. 9EveneFAHeptadecanoic acidLARODANC17: 0C17H34O2270. 31327. 324. 5OddoFANonadecanoic acidLARODANC19: 0C19H38O2298. 31355. 326. 9OddoFA9Z-Hexadecenoic acidLARODANC16: 1n-7C16H30O2254. 21311. 222. 6MonounsaturatedMUFA10Z-octadecenoic acidTentatively assignedC18: 1C18H34O2282. 31339. 325. 6MonounsaturatedMUFA9Z-octadecenoic acidLARODANC18: 1n-9C18H34O2282. 31339. 325. 5MonounsaturatedMUFA9Z, 12Z-Octadecadienoic acidLARODANC18: 2C18H32O2280. 21337. 225. 4PolyunsaturatedPUFACholest-5-en-3β-olTRCCholesterolC27H46O386. 61458. 136. 6SterolCholCholest-5, 24-dien-3β-olTRCDesmosterolC27H44O384. 61456. 137. 0SterolSterold17PalmitateC/D/N Isotopesd17C16: 0C16H15D17O2273. 51330. 522. 7EveneFAd6CholesterolC/D/N Isotopesd6CholesterolC27H40D6O392. 71464. 436. 5SterolChold6DesmosterolTRCd6DesmosterolC27H38D6O390. 71462. 236. 9SterolSterold6SqualeneTRCd6SqualeneC30H44D6416. 8075. 433. 2PrenolSqn-hexadecyl-1, 1, 2, 2-d4 hexadecanoate-16, 16, 16-d3C/D/N Isotopesd7WE 32: 0C32H57D7O2487. 90487. 938. 8Wax esterWE1, 2, 3-PropanetriolTentatively assignedGlycerolC3H8O392. 13205. 08. 6GlycerolGly (2S) -5-oxopyrrolidine-2-carboxylic acidTentatively assigned5-OxoprolineC5H7NO3129. 12156. 113. 6Amino acidAAβ-D-Fructofuranosyl α-D-glucopyranosideTentatively assignedSucroseC12H22O11342. 38361. 331. 1SugarSUnknown193Tentatively assignedUnknown193UnUnUn193. 116. 0UnUnUnknown296Tentatively assignedUnknown296UnUnUn296. 115. 9UnUnlanosta-8, 24-dien-3β-olTentatively assignedLanosterolC30H50O426. 71393. 238. 5SterolSterolcholest-7-en-3β-olTentatively assignedLathosterolC27H46O386. 61458. 137. 2SterolSterol5-α-Cholesta-8, 24-dien-3-β-olTentatively assignedZymosterolC27H44O384. 61456. 137. 6SterolSterolCarbamideTentatively assignedUreaCH4N2O60. 02189. 28. 3AmideUAbbreviations: AA, amino acid; Avg, average; d7WE, n-hexadecyl-1, 1, 2, 2-d4 hexadecanoate-16, 16, 16-d3; GC-MS, gas chromatography–mass spectrometry; FA, fatty acid, MUFA, monounsaturated fatty acid; MW, molecular weight; PUFA, polyunsaturated fatty acid; RT, retention time (min) ; TMS, trimethyl silyl group; TRC, Toronto Research Chemicals; Un, unknown; WE, wax ester. The m/z ratio in bold were taken into account for the quantitative assessments. Open table in a new tab Abbreviations: AKT, protein kinase B; IVL, involucrin; K, keratin; LOR, loricrin; p-AKT, phosphorylated protein kinase B; PCNA, proliferating cell nuclear antigen; p-ERBB, phosphorylated ERBB; p-EGFR, phosphorylated EGFR; p-P44/42 MAPK, phosphorylated P44/42 MAPK; VCL, vinculin. Abbreviations: AA, amino acid; Avg, average; d7WE, n-hexadecyl-1, 1, 2, 2-d4 hexadecanoate-16, 16, 16-d3; GC-MS, gas chromatography–mass spectrometry; FA, fatty acid, MUFA, monounsaturated fatty acid; MW, molecular weight; PUFA, polyunsaturated fatty acid; RT, retention time (min) ; TMS, trimethyl silyl group; TRC, Toronto Research Chemicals; Un, unknown; WE, wax ester. The m/z ratio in bold were taken into account for the quantitative assessments. Mice carrying floxed Erbb2 alleles or Erbb3 alleles or expressing cre recombinase under the keratin 5 promoter (a courtesy of A. Ramirez and J. Jorcano, CIEMAT, Madrid, Spain) have been described previously (Garratt et al. , 2000Garratt A. N. Voiculescu O. Topilko P. Charnay P. Birchmeier C. A dual role of erbB2 in myelination and in expansion of the schwann cell precursor pool. J Cell Biol. 2000; 148: 1035-1046Crossref PubMed Scopus (231) Google Scholar; Lee et al. , 2009Lee D. Yu M. Lee E. Kim H. Yang Y. Kim K. et al. Tumor-specific apoptosis caused by deletion of the ERBB3 pseudo-kinase in mouse intestinal epithelium. J Clin Invest. 2009; 119: 2702-2713Crossref PubMed Scopus (78) Google Scholar; Ramirez et al. , 2004Ramirez A. Page A. Gandarillas A. Zanet J. Pibre S. Vidal M. et al. A keratin K5Cre transgenic line appropriate for tissue-specific or generalized Cre-mediated recombination. Genesis. 2004; 39: 52-57Crossref PubMed Scopus (166) Google Scholar). Mice strains were maintained in the C57BL/6N background under specific pathogen-free conditions and had access to water and standard rodent diet (V1534, Ssniff) ad libitum. All animal experiments were approved by the governmental body of the state of Upper Bavaria, Germany (Regierung von Oberbayern, 55. 2-1-54-2532-206-2012) and by the Austrian Federal Ministry of Education, Science, and Research (BMBWF-68. 205/106-V/3b/2019) and were performed in strict compliance with the European Communities Council Directive (86/609/EEC) recommendations for the care and use of laboratory animals. Genotyping of mouse lines was performed according to the original publications. Both females and males of E2/E3del double knockouts show the same phenotype, but all experiments published in this article were performed with males. HaCaT cells were originally obtained from CLS Cell Lines Service. For the generation of ERBB2 and ERBB3 double-knockout clones, we used the already established ERBB2-knockout cell line HaCaT 2/F6 and the ERBB3-knockout cell line HaCaT 3/B8 (Dahlhoff et al. , 2017Dahlhoff M. Gaborit N. Bultmann S. Leonhardt H. Yarden Y. Schneider M. R. CRISPR-assisted receptor deletion reveals distinct roles for ERBB2 and ERBB3 in skin keratinocytes. FEBS J. 2017; 284: 3339-3349Crossref PubMed Scopus (0) Google Scholar). Cells were cultured in DMEM high glucose (Merck) supplemented with 10% fetal calf serum (Merck) and 1% streptomycin/penicillin (Thermo Fisher Scientific) in a humidified incubator at 37 °C with 5% carbon dioxide. The following plasmids were used for the CRISPR/Cas9-based deletion of ERBB2 and ERBB3: Cas9 plasmid (Mali et al. , 2013Mali P. Yang L. Esvelt K. M. Aach J. Guell M. DiCarlo J. E. et al. RNA-guided human genome engineering via Cas9. Science. 2013; 339: 823-826Crossref PubMed Scopus (7279) Google Scholar), surrogate plasmid, and guide RNA (gRNA) plasmid (Mulholland et al. , 2015Mulholland C. B. Smets M. Schmidtmann E. Leidescher S. Markaki Y. Hofweber M. et al. A modular open platform for systematic functional studies under physiological conditions. Nucleic Acids Res. 2015; 43: e112Crossref PubMed Scopus (0) Google Scholar) with the gRNA sequences 5′-CTGGACATGCTCCGCCACCTCTACCA-3′ for ERBB2 gRNA and 5′-TACGAGAGGTGTGAGGTGGTGATG-3′ for ERBB3 gRNA. For transfection, we used Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Selection of cells with Cas9 activity was accomplished by single-cell sorting for GFP and mCherry-positive cells 48 hours after transfection. The cells were dissociated with 0. 25% Trypsin-EDTA (Merck), resuspended in DMEM containing 2% fetal calf serum, and sorted using CytoFLEX SRT (Beckman Coulter) into 96-well plates (Falcon) containing 100 μl DMEM complete medium. After expansion of the clones, the sequence variation was analyzed by PCR amplification of the gRNA-binding region using the following primers: 5′-TCTCCCTGTCTGAGGTGGC-3′ for hERBB2ₛ, 5′-GGGACATGATCATGCTGGC-3′ for hERBB2ₐs, 5′-CTACAGCTTCTGCCTATCGC-3′ for hERBB3ₛ, and 5′-TAGGTCCCAGATGACAGCC-3′ for hERBB3ₐs. PCR products were purified by gel extraction (New England Biolabs) and cloned using the StrataClone Blunt PCR Cloning Kit (Agilent Technologies). Six different clones were sequenced using T7 primer 5′-TAATACGACTCACTATAGGG-3′. Identified knockouts were confirmed by western blot analysis. Normal human epidermal keratinocytes were isolated from healthy human skin biopsies. The dermis was removed as good as possible, and the remaining tissue was digested overnight at 4 °C using Dispase II (2. 2 IU/ml, Roche). Subsequently, the epidermis was separated and incubated with Trypsin/EDTA (Lonza) for 5 minutes at 37 °C to isolate primary keratinocytes. Normal human epidermal keratinocytes were cultured in Keratinocyte Growth Medium 2 (PromoCell) supplemented with 1% streptomycin/penicillin (Thermo Fisher Scientific) in a humidified incubator at 37 °C with 5% carbon dioxide. Cells were passaged approximately twice a week depending on confluency. Lipofectamine RNAiMAX (Invitrogen) was used according to the manufacturer's instructions to transfect normal human epidermal keratinocytes at a confluence of 50–60% in a 6-well plate with 30 pmol pooled small interfering RNAs for ERBB2 and ERBB3 or with a negative control small interfering RNA (Silencer Select, Ambion). After 96 or 144 hours, cells were harvested with ice-cold PBS for protein isolation. Protein was extracted using RIPA lysis buffer with protease inhibitor (Merck) and PhosSTOP (Roche). The concentration of the protein lysates was estimated by bicinchoninic acid assay (Thermo Fisher Scientific). A total of 20 μg of HaCaT total protein or 10 μg of normal human epidermal keratinocyte total protein was separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (GE Healthcare) by semidry blotting for 1 hour at 14 V. The membranes were stained with Ponceau S solution to confirm successful blotting and then blocked in 5% w/v skim milk for 1 hour at room temperature. After washing in Tris-buffered saline (TBS) solution with 0. 05% Tween 20 (Merck), membranes were incubated overnight at 4 °C in 5% w/v BSA (Sigma-Aldrich) with the appropriate primary antibody. Antibodies and their dilutions are listed in Supplementary Table S1. Membranes were washed and incubated in 5% w/v skim milk powder with the corresponding horseradish peroxidase–labeled secondary antibody. Signals were detected using an enhanced chemiluminescence detection reagent (Bio-Rad Laboratories). After detection, membranes were stripped by incubation with an appropriate buffer (2% SDS, 62. 5 mM Tris/hydrogen chloride, pH 6. 8, and 100 mM β-mercaptoethanol) for 40 minutes at 60 °C and incubated with a second primary antibody recognizing the total protein of a phosphorylated protein or a housekeeper protein. Band density measurement was performed using ImageJ (http: //rsb. info. nih. gov/ij), and values were normalized to GAPDH. Cell proliferation and migration were monitored in 6-well plates using a PHIO Cellwatcher M microscope (PHIO Scientific, Munich, Germany) placed inside the incubator, imaging each well every 30 minutes (over a period of x to y hours). The proliferation, motility, and wound healing assays were analysed using PHIOme Data Management and Analysis Platform with the Software Add-Ons Proliferation, Motility, and Wound Healing, version 1. 4. 2, from PHIO Scientific. For the proliferation assay, 8 × 104 HaCaT cells, either ERBB2 or ERBB3 single- or double-knockout cells and a mock-transfected control cell line, were seeded in a 6-well plate. The cells were allowed to attach for 24 hours, and then they were washed twice with PBS and covered with 3 ml culture medium, and then the plate was transferred to the PHIO Cellwatcher. Migration was assessed by wound healing assays. Therefore, cells were seeded on a 6-well plate and allowed to attach and grow until they were 100% confluent. The cell monolayer was scraped with a 200-μl pipet tip. To remove the debris, the scratch was washed twice with PBS before adding 3 ml of culture medium and starting the measurement. A confluency experiment was performed to study the ERBB receptors under conditions of high and low cell–cell contact. Briefly, the cells were seeded and cultured up to 50 or 100% confluency. Then, they were harvested using RIPA lysis buffer. Skin samples were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. For target retrieval, the sections were boiled in a microwave for 20 minutes in 10 mM citrate buffer (pH 6. 0). Tissue sections were incubated with an anti–Ki-67 antibody (rat anti-mouse Ki-67, TEC-3, DakoCytomation, 1: 200) over night at 4 °C, followed by incubation with a horseradish peroxidase–conjugated secondary antibody rabbit anti-rat antibody (DakoCytomation, E0468, 1: 200) for 1 hour at room temperature. The compound 3, 3'-diaminobenzidine (KemEnTec, Copenhagen, Denmark) was used as a chromogen. Tumor cell proliferation rate was measured on Ki-67–stained sections by counting 10 visual fields for each mouse. Both Ki-67–positive and –negative nuclei were counted on pictures taken with a 200-magnification lens and a Leica DFC425C digital camera (Leica Microsystems) covering an area of 1. 3 mm2. Skin samples were fixed in 4% paraformaldehyde in PBS, dehydrated, and embedded in paraffin. Paraformaldehyde-fixed sections were microwaved for 22 minutes in 10 mM citrate buffer (pH 6. 0). Tissue sections were blocked with 5% normal donkey serum in TBS for 1 hour at room temperature and incubated over night at 4 °C with the designated primary antibodies in 1% BSA in TBS. Afterward, the slides were washed with TBS with 0. 1% Tween and incubated with the appropriate fluorescent secondary antibody in 1% BSA in TBS for 1 hour at temperature. After washing with TBS, the sections were mounted with Vectashield with DAPI (Vectorlabs). Images were acquired using Zeiss LSM 880 Airyscan confocal laser-scanning microscope (Carl Zeiss MicroImaging) with a ×40/1. 4